The maintenance of polarized plasma membrane domains with distinct protein and lipid compositions is critical for efficient renal cell function. The distribution of receptors and ion transporters in these cells is regulated in part by the rate at which these proteins are internalized from the apical and basolateral cell surfaces. The mechanisms that underlie cargo recruitment to clathrin coated pits where this process is initiated are poorly understood. Localized synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) at endocytic sites plays multiple roles in endocytosis, and modulation of surface PIP2 levels in nonpolarized cells can differentially affect the internalization of distinct proteins. The central hypothesis of this proposal is that apical and basolateral PIP2 metabolism in renal epithelial cells is independently regulated to selectively modulate clathrin-dependent endocytosis from these domains. Consistent with this idea, that the three isoforms of PI5-kinase (the enzymes that generate cell surface PIP2) are differentially localized in polarized renal epithelial cells. The specific aims of our proposal are to determine whether apical and basolateral pools of PIP2 are independently modulated in renal epithelial cells;to examine how changes in PIP2 levels upon agonist stimulation of G protein coupled receptors affect endocytosis from polarized membrane domains;and to determine how the apically localized PI5-kinase mPI5KI1 regulates apical endocytosis. The results of our studies have important implications for our understanding of how polarized cells compartmentalize PI synthesis and catabolism and for how this process is affected during normal signaling and in renal disease. PUBLIC HEALTH RELEVANCE: The surface of kidney cells is segregated into distinct domains with unique protein and lipid compositions that face the urine and the bloodstream. This asymmetric distribution of surface components is essential for kidney cells to properly reabsorb water, ions, and metabolites from the forming urine. These processes are controlled by receptors and ion transporters whose delivery to and removal from the cell surface is regulated by numerous cellular factors. Our goal is to understand how kidney cells differentially regulate the removal of these proteins from the surface, and how these mechanisms are selectively adjusted during the response to specific physiological signals. The results of our research will provide new information about how the trafficking of kidney proteins is regulated under normal conditions and under conditions of physiological stress.